Treatment of t-cell mediated immune disorders

ABSTRACT

A method for suppressing T cell activation which comprises contacting a cell population comprising T cells in vitro or ex vivo with an effective amount of STRO-1 +  cells and/or soluble factors derived therefrom to suppress T cell activation.

FIELD

The present disclosure provides methods and compositions for suppressingT cell activation ex vivo or in vivo. The methods and compositions areuseful in the treatment of disorders caused by excessive or aberrant Tcell activation, such as autoimmune diseases.

BACKGROUND

The CD4⁺ T-lymphocyte (herein referred to as the CD4⁺ T-cell) is thecentral player in the immune system because of the “help” it provides toother leukocytes in fighting off infection and potential cancerouscells. CD4⁺ T-cells play essential roles in both humoral andcell-mediated immunity and additionally they act during parasiteinfection to promote the differentiation of eosinophils and mast cells.If the CD4⁺ T-cell population is deleted (as is the case in AIDSpatients) the host is rendered susceptible to a number of pathogens andtumors that do not ordinarily pose a threat to the host.

While CD4⁺ T-cells thus play an important beneficial role in diseaseprevention, the aberrant function of these cells can produce seriousproblems. In some individuals, the aberrant function of CD4⁺ T-cellsleads to autoimmunity and other disease states. Autoimmune diseases inwhich CD4⁺ T-cells have been implicated include multiple sclerosis,rheumatoid arthritis and autoimmune uveitis. In essence these diseasesinvolve an aberrant immune response in which the immune system issubverted from its normal role of attacking invading pathogens andinstead attacks the host body tissues, leading to illness and evendeath. The targeted host tissues vary between autoimmune diseases, forexample, in multiple sclerosis the immune system attacks the whitematter of the brain and spinal cord, in rheumatoid arthritis the immunesystem attacks the synovial lining of the joints. Activated CD4⁺ T-cellshave also been implicated in other illnesses, including rejection oftransplant tissues and organs and in the development of CD4⁺ T-celllymphomas.

Investigations into conditions caused by aberrant CD4⁺ T-cells activityarc focussed on several animal models, and in particular on a number ofexperimentally induced autoimmune diseases. Research on theseexperimentally induced diseases in animals is premised on the idea thatthey will provide information useful in the treatment of thecorresponding human diseases. In pursuit of this goal, it has been shownthat CD4⁺ T-cells arc responsible for several experimentally inducedautoimmune diseases in animals, including experimental autoimmuneencephalomyelitis (EAE), collagen induced arthritis (CIA), andexperimental autoimmune uveitis (EAU).

EAE is induced by autoimmunizing animals against myelin basic protein(MBP, component of the white matter of the brain and the spinal cord)and produces the same clinical symptoms observed in multiple sclerosis:demyelination and paralysis. Proof of the value of the EAE model as acomparative model for multiple sclerosis has been provided by evidenceshowing that these conditions share a causative nexus: Steinman andco-workers showed that the predominant cell type found in the brainlesions of multiple sclerosis patients is CD4⁺ T-cells (Oksenberg etal., 1990, Nature 345:344-345) and that the T-cell receptor (themolecule responsible for antigen recognition) associated with the cellsin these brain lesions had the same 3 amino acid binding motif forantigen recognition as on the CD4⁺ T-cells responsible for causingexperimental autoimmune encephalomyelitis (EAE) (Oksenberg et al., 1993,Nature 362:68-70). The evidence thus suggests that the EAE model will beuseful in testing therapies for disorders caused by aberrant CD4⁺ T-cellactivity.

While it appears that therapeutic approaches that destroy the CD4⁺T-cell population might be effective in ameliorating these autoimmunediseases, this approach has one very major drawback. The treatment notonly destroys those CD4⁺ T-cells that are antigen reactive and thusinvolved in the autoimmune disease process, but also the CD4⁺ T-cellsthat are quiescent and not involved in the disease. Since CD4⁺ T-cellsare important in the general immune response (protecting the bodyagainst infectious agents), destruction of the entire CD4⁺ T-cellspopulation leaves the patient severely immunocompromised and hencehighly susceptible to infection. A preferable approach would be tosuppress activation of CD4⁺ T-cells in cases of excessive or aberrantCD4⁺ T-cell activity.

SUMMARY

The present disclosure provides a method for suppressing T cellactivation which comprises contacting a cell population comprising Tcells in vitro or ex vivo with an effective amount of STRO-1⁺ cellsand/or soluble factors derived therefrom to suppress T cell activation.

In one example the STRO-1⁺ cells and/or soluble factors derivedtherefrom suppress T cell receptor activation.

In one example the cell population is a peripheral blood mononuclearcell sample.

In another example the cell population comprise CD25⁺ CD4⁺ T cells of anaive phenotype (CD45RA⁺).

In another example the method comprises contacting the cell populationcomprising T cells in vitro or ex vivo with an effective amount ofSTRO-1⁺ cells and/or soluble factors derived therefrom and one or morefactors which induce formation of regulatory T cells.

The one or more factors which induce formation of regulatory T cells maybe selected from the group consisting of α-melanocyte-stimulatinghormone (α-MSH), transforming growth factor-β2 (TGF-β2), vitamin D3and/or Dexamethasone.

In another example the method further comprises contacting the cellpopulation comprising T cells with one or more agents selected from thegroup consisting of interleukins, antigens, antigen presenting cells,lectins, and antibodies or specific ligands for a cell surface receptorsor combinations thereof.

The interleukin may be selected from the group consisting of IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18 or combinations thereof.

The present disclosure also provides a composition of T cells obtainedby a method described herein.

The present disclosure also provides a composition comprising T cells,STRO-1⁺ cells and/or soluble factors derived therefrom, one or morefactors which induce formation of regulatory T cells and apharmaceutically acceptable carrier.

The present disclosure also provides a method for treating an autoimmunedisorder in a subject in need thereof comprising treating a cellpopulation comprising T cells in vitro or ex vivo with an effectiveamount of STRO-1⁺ cells and/or soluble factors derived therefrom tosuppress T cell activation and administering the treated cells to thesubject.

The present disclosure also provides a method for treating or preventinga disorder caused by excessive or aberrant T cell activation comprisingadministering to a subject in need thereof an amount of STRO-1⁺ cellsand/or soluble factors derived therefrom effective to suppress T-cellactivation in the patient.

The method may further comprise administering to the subject one or moreagents selected from the group consisting of interleukins, antigens,antigen presenting cells, lectins, and antibodies or specific ligandsfor a cell surface receptors or combinations thereof. The interleukinmay be selected from the group consisting of IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18 or combinations thereof.

Additional active agents that may be conjointly administered withtreated T cells or STRO-1⁺ cells and/or soluble factors derivedtherefrom include, but are not limited to, beta-interferons,corticosteroids, non-steroid anti-inflammatory drugs, tumor necrosisalpha blockers, antimalarial drugs, cyclosporines, tumor necrosis alphainhibitors, immunosuppressants, immunomodulators, antibody therapeutics,cell-based therapies and T cell epitopes (e.g., ToleroTrans TransplantRejection Therapy by Circassia, etc.).

In some examples the Stro-1⁺ cells and/or progeny cells thereof and/orsoluble factors derived therefrom are genetically engineered to expressa molecule to block co-stimulation of T-cells.

In some examples of the methods of the disclosure the STRO-1⁺ cells areenriched for STRO-1^(bright) cells.

The STRO-1⁺ cells may be autogeneic or allogeneic. In one example, theSTRO-1^(bright) cells are allogeneic.

In another example of this method, the STRO-1⁺ cells and/or progenycells thereof have been expanded in culture prior to obtaining thesoluble factors.

In another example of this method the STRO-1⁺ cells and/or progeny cellsthereof are administered in a dosage ranging from 10⁵ to 10¹⁰ cells.

Exemplary dosages of the cells include between 0.1×10⁶ to 5×10⁶ STRO-1⁺cells and/or progeny thereof. For example, the method comprisesadministering between 0.3×10⁶ to 2×10⁶ STRO-1⁺ cells and/or progenythereof.

One form of the method involves administering a low dose of STRO-1⁺cells and/or progeny thereof. Such a low dose is, for example, between0.1×10⁵ and 0.5×10⁶ STRO-1⁺ cells and/or progeny thereof, such as about0.3×10⁶ STRO-1⁺ cells and/or progeny thereof.

The present disclosure also contemplates numerous administrations of thecells and/or soluble factors. For example, such a method can involveadministering the cells and monitoring the subject to determine when oneor more symptoms of an autoimmune disorder occurs or recurs andadministering a further dose of the cells and/or soluble factors.Suitable methods for assessing symptoms of autoimmune disorders will beapparent to the skilled artisan and/or described herein.

In one example, the population enriched for STRO-1⁺ cells and/or progenythereof and/or soluble factors derived therefrom are administered onceweekly or less often, such as, once every four weeks or less often.

In another embodiment, the population of cells enriched forSTRO-1^(bright) cells and/or progeny cells thereof and/or solublefactors derived therefrom is administered systemically. For example, thepopulation of cells enriched for STRO-1^(bright) cells and/or progenycells thereof and/or soluble factors derived therefrom may beadministered intravenously, intra-arterially, intramuscularly,subcutaneously, into an aorta, into an atrium or ventricle of the heartor into a blood vessel connected to an organ, e.g., an abdominal aorta,a superior mesenteric artery, a pancreaticoduodenal artery or a splenicartery.

In another example the methods of the disclosure further compriseadministering an immunosuppressive agent. The immunosuppressive agentmay be administered for a time sufficient to permit said transplantedcells to be functional. In one example, the immunosuppressive agent iscyclosporine. The cyclosporine may be administered at a dosage of from 5to 40 mg/kg body wt.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Co-expression of TNAP (STRO-3) and the Mesenchymal PrecursorCell Marker, STRO-1^(bright) by Adult Human BMMNC. Dual-colorimmunofluorescence and flow cytometry was performed by incubation ofSTRO-1 MACS-selected BMMNC and indirectly labelled with a goatanti-murine IgM antibody coupled to FITC (x axis), and STRO-3 mAb(murine IgG1) indirectly labelled with a goat anti-murine IgG coupled toPE (y axis). The dot plot histogram represents 5×104 events collected aslistmode data. The vertical and horizontal lines were set to thereactivity levels of <1.0% mean fluorescence obtained with theisotype-matched control antibodies, 1B5 (IgG) and 1A6.12 (IgM) treatedunder the same conditions. The results demonstrate that a minorpopulation of STRO-1^(bright) cells co-expressed TNAP (upper rightquadrant) while the remaining STRO-1+ cells failed to react with theSTRO-3 mAb.

FIG. 2. Gene expression profile of STRO-1^(bright) or STRO-1^(dim)progeny of cultured and expanded STRO-1^(bright) MPC. Single cellsuspensions of ex vivo expanded bone marrow MPC were prepared bytrypsin/EDTA treatment. Cells were stained with the STRO-1 antibodywhich was subsequently revealed by incubation with goat-anti murineIgM-fluorescein isothiocyanate. Total cellular RNA was prepared frompurified populations of STRO-1^(dim) or STRO-1^(bright) expressingcells, following fluorescence activated cell sorting (A). Using RNAzolBextraction method, and standard procedures, total RNA was isolated fromeach subpopulation and used as a template for cDNA synthesis. Theexpression of various transcripts was assessed by PCR amplification,using a standard protocol as described previously (Gronthos et al. JCell Sci. 116:1827-1835, 2003). Primers sets used in this study areshown in Table 2. Following amplification, each reaction mixture wasanalysed by 1.5% agarose gel electrophoresis, and visualised by ethidiumbromide staining (B). Relative gene expression for each cell marker wasassessed with reference to the expression of the house-keeping gene,GAPDH, using ImageQant software (C).

FIG. 3. STRO-1^(bright) progeny of cultured and expanded STRO-1⁺ MPCexpress high levels of SDF-1, STRO-1^(dim) progeny do not. (A)MACS-isolated preparations of STRO-1⁺ BMMNCs were partitioned intodifferent STRO-1 subsets according to the regions, STRO-1^(bright) andSTRO-1^(dim/dull) using FACS. Total RNA was prepared from each STRO-1subpopulation and used to construct a STRO-1^(bright) subtractionhybridization library (B-C). Replicate nitrocellulose filters, whichhave been blotted with representative PCR products amplified frombacterial clones transformed with STRO-1^(bright) subtracted cDNA. Thefilters were then probed with either [³²P] deoxycytidine triphosphate(dCTP)—labeled STRO-1^(bright) (B) or STRO-1^(dim/dull) (C) subtractedcDNA. The arrows indicate differential expression of 1 clone containinga cDNA fragment corresponding to human SDF-1. (D) Reverse transcriptase(RT)—PCR analysis demonstrating the relative expression of SDF-1 andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts in totalRNA prepared from freshly MACS/FACS-isolated BMMNC STRO-1 populationsprior to culture. bp indicates base pair.

FIG. 4. Comparative efficiency of STRO-1 negative MSC (preparation A)and STRO-1^(bright) MPC (preparation B) for inhibition of T cellproliferation. PBMC were stimulated with CD3/CD28 coated beads for 4days in the absence or presence of preparations A or B. T cellproliferation was measured by ³H-Tdr incorporation as counts per minute(cpm).

FIG. 5. Comparative efficiency of STRO-1 negative MSC (preparation A)and STRO-1^(bright) MPC (preparation B) for inhibition of T cellproliferation. PBMC were stimulated with CD3/CD28 coated beads for 4days in the presence of different concentrations of preparations A or B.T cell proliferation in the various cultures were was measured by ³H-Tdrincorporation and reported as percentage of the control T cellproliferation in which PBMC were stimulated in the absence of MSC.

FIG. 6. STRO-1^(bright) MPC reduce or prevent T cell immune response toa specific antigen. Splenocytes were obtained from MPC treated mice andcontrols on day 36 after MOG₃₅₋₅₅ immunization. Splenocytes werecultured in vitro and restimulated with MOG₃₅₋₅₅ and T-cellproliferative responses were measured through [³H]-thymidineincorporation.

FIG. 7. STRO-3 immunoselected and culture expanded human MPCs inhibitPHA activation of T cells. PMBC were stimulated with phytohemagglutinin(PHA) to illicit lymphocyte proliferation. STRO-3 selected MPCs wereable to significantly suppress PMBC T-cell proliferation over a range ofconcentrations.

FIG. 8. STRO-3 immunoselected and culture expanded ovine STRO-1^(bright)cells (MPCs) induce very low levels of alloimmune responses, and inhibitPHA activation of T cells. Ovine MPCs did not directly induce lymphcyteproliferation when used at a stimulator cell at 1%, 5%, 10% 20% or 50%dilutions and were able to significantly inhibit levels of alloimmuneresponses to ovine PBMCs stimulated with PHA.

FIG. 9. Dose dependent immunosuppressive effects of ovine STRO-3immunoselected and culture expanded STRO-1^(bright) cells (MPCs) onPHA-mediated lymphocyte proliferation. Ovine MPCs were able to suppresslymphocyte proliferation in a dose dependent manner when used asstimulators against ovine PBMCs or purified ovine T cells (selectedusing Miltenyi T cell isolation kit).

DETAILED DESCRIPTION

The present disclosure demonstrates that STRO-1⁺ cells inhibit T cellactivation via antigen and non-antigen specific mechanisms. For example,the present disclosure demonstrates that STRO-1⁺ cells inhibitMOG-specific T cell proliferative responses in vivo. The presentdisclosure also demonstrates that STRO-1⁺ cells inhibit anti-CD3mediated T cell proliferative responses in vitro. This disclosuretherefore provides new therapeutic approaches for treating or preventingdiseases where aberrations in regulatory T cell number and/or functionhave been observed (e.g., in autoimmune disorders).

Definitions

As used herein, the terms “treatment”, “treating”, and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disorder or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disorder (e.g., autoimmunedisease) and/or adverse affect attributable to the disorder.“Treatment”, as used herein, covers any treatment of a disease in amammal, particularly in a human, and includes: (a) increasing survivaltime; (b) decreasing the risk of death due to the disease; (c)preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;(d) inhibiting the disease, i.e., arresting its development (e.g.,reducing the rate of disease progression); and (e) relieving thedisease, i.e., causing regression of the disease.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample.

As used herein the terms “subject” and “patient” refer to animalsincluding mammals, including humans. The term “mammal” includesprimates, domesticated animals including dogs, cats, sheep, cattle,horses, goats, pigs, mice, rats, rabbits, guinea pigs, captive animalssuch as zoo animals, and wild animals.

Treatment Methods

The present disclosure provides methods for suppressing T cellactivation that can be effected in vitro or in vivo.

In some embodiments the method for suppressing T cell activationcomprises contacting a cell population comprising T cells in vitro or exvivo with an effective amount of STRO-1⁺ cells and/or soluble factorsderived therefrom for a period of time sufficient to suppress T cellactivation.

In some embodiments, the method comprises obtaining a cell populationthat comprises T cells (e.g., CD4⁺ cells) and contacting the T cellswith STRO-1⁺ cells and/or soluble factors derived therefrom for a periodof time sufficient to suppress T cell receptor activation.

In one embodiment the STRO-1⁺ cells and/or soluble factors derivedtherefrom stimulate formation or expansion of regulatory T cells withinthe cell population. Regulatory T cells are a subset of T cells thatsuppress the activity of effector T cells and are characterized by themarkers CD4⁺ CD25⁺. In some embodiments, the regulatory T cells areFoxP3⁺ and/or IL-10 producing regulatory T cells.

Accordingly, in a further embodiment the method of the disclosurecomprises culturing the cell population comprising T cells in vitro orex vivo with an effective amount of STRO-1⁺ cells and/or soluble factorsderived therefrom and one or more factors that stimulate formation ofregulatory T cells, such as a-melanocyte-stimulating hormone (α-MSH)and/or transforming growth factor-β2 (TGF-β2). In some aspects, theculture also contain vitamin D3 and/or Dexamethasone, which havedemonstrated to promote the generation of IL-10-producing regulatory Tcells (Barrat et al. J. Exp. Med. 195(5): 2002, 603-616).

In some embodiments, the T cells are isolated from a mammalian sampleprior to exposure to STRO-1⁺ cells and/or soluble factors derivedtherefrom.

The term “isolated” with respect to T cells refers to cell populationpreparation in a form that has at least 70, 80, 90, 95, 99, or 100% Tcells. In some aspects, a desired cell population is isolated from othercellular components, in some instances to specifically exclude othercell types that may “contaminate” or interfere with the study of thecells in isolation. It is to be understood, however, that such an“isolated” cell population may incorporate additional cell types thatare necessary for cell survival or to achieve the desired resultsprovided by the disclosure. For example, antigen presenting cells, suchas monocytes (macrophages) or dendritic cells, may be present in an“isolated” cell population of T cells or added to a population ofisolated T cells for generation of regulatory T cells. In some aspects,these antigen presenting cells may be activated monocytes or dendriticcells. Cell populations comprising T cells for use in the methods of thedisclosure may be isolated from a biological sample taken from amammalian subject. The sample may originate from a number of sources,including, but not limited to peripheral blood, leukapheresis bloodproduct, apheresis blood product, bone marrow, thymus, tissue biopsy,tumor, lymph node tissue, gut associated lymphoid tissue, mucosaassociated lymphoid tissue, liver, sites of immunologic lesions (e.g.,synovial fluid), pancreas, and cerebrospinal fluid. The donor subject ispreferably human, and can be fetal, neonatal, child, adult, and may benormal, diseased, or susceptible to a disease of interest.

In some embodiments, the T cell sample comprises peripheral bloodmononuclear cells

(PBMCs) from a blood sample. By “peripheral blood mononuclear cells” or“PBMCs” is meant lymphocytes (including T-cells, B-cells, NK cells,etc.) and monocytes. In general, PBMCs are isolated from a patient usingstandard techniques. In some embodiments, only PBMCs are taken, eitherleaving or returning substantially all of the red blood cells andpolymorphonuclear leukocytes to the donor. PBMCs may be isolated usingmethods known in the art, such as leukophoresis. In general, a 5 to 7liter leukophoresis step is performed, which essentially removes PBMCsfrom a patient, returning the remaining blood components. Collection ofthe sample is preferably performed in the presence of an anticoagulant(e.g., heparin).

The T cell-containing sample comprising PBMCs or isolated T cells can bepretreated using various methods before treatment with STRO-1⁺ cellsand/or soluble factors derived therefrom. Generally, once collected, thecells can be additionally concentrated, if this was not donesimultaneously with collection or to further purify and/or concentratethe cells. For example, PBMCs can be partially purified by densitygradient centrifugation (e.g., through a Ficoll-Hypaque gradient). Cellsisolated from a donor sample are normally washed to remove serumproteins and soluble blood components, such as autoantibodies,inhibitors, etc., using techniques well known in the art. Generally,this involves addition of physiological media or buffer, followed bycentrifugation. This may be repeated as necessary. The cells can then becounted, and in general, from 1×10⁹ to 2×10⁹ white blood cells arecollected from a 5-7 liter leukapheresis. The purified cells can beresuspended in suitable media or buffer to maintain viability. Suitablesolutions for resuspension will generally be a balanced salt solution(e.g., normal saline, PBS, Hank's balanced salt solution, etc.)optionally supplemented with fetal calf serum, BSA, HSA, normal goatserum, and/or other naturally occurring factors, in conjunction with anacceptable buffer at low concentration, generally from 5-50 mM.Convenient buffers include, but are not limited to HEPES, phosphatebuffers, lactate buffers, etc.

A specific cell type (e.g., effector T cells, regulatory T cells, etc.)can be separated from a complex mixture of cells using techniques thatenrich for cells having the desired characteristic (e.g., CD4⁺, FoxP3⁺,etc.). Most standard separation methods use affinity purificationtechniques to obtain a substantially isolated cell population.Techniques for affinity separation may include, but are not limited to,magnetic separation (e.g., using antibody-coated magnetic beads),affinity chromatography, cytotoxic agents joined to a monoclonalantibody (e.g., complement and cytotoxins), and “panning” with antibodyattached to a solid matrix. Techniques providing accurate separationinclude fluorescence activated cell sorting, which can have varyingdegrees of sophistication, such as multiple color channels, impedancechannels, etc. The living cells may be selected against dead cells byemploying dyes that associate with dead cells (e.g., propidium iodide,LDS, etc.). Any technique may be used that is not unduly detrimental tothe viability of the selected cells.

The affinity reagents used may be specific receptors or ligands for cellsurface molecules (e.g., CD4, CD25, etc.). Antibodies may be monoclonalor polyclonal and may be produced by transgenic animals, immunizedanimals, immortalized B-cells, and cells transfected with DNA vectorsencoding the antibody. Details of the preparation of antibodies andtheir suitability for use as specified binding members are well-known tothose skilled in the art. In addition to antibody reagents, peptide-MHCantigen and T cell receptor pairs may be used, as well as peptideligands, effector and receptor molecules.

Antibodies used as affinity reagents for purification are generallyconjugated with a label for use in separation. Labels may includemagnetic beads (which allow for direct separation), biotin (which can beremoved with avidin or streptavidin bound to a support), fluorochromes(which can be used with a fluorescence activated cell sorter), or othersuch labels that allow for ease of separation of the particular celltype. Fluorochromes may include phycobiliproteins, such as phycoerythrinand allophycocyanins, fluorescein and Texas red. Frequently, eachantibody is labeled with a different fluorochrome to permit independentsorting for each marker.

For purification of a desired cell population, cell-specific antibodiesare added to a suspension of cells and incubated for a period of timesufficient to bind the available cell surface antigens. The incubationwill usually be at least about 5 minutes and usually less than about 30minutes. It is desirable to have a sufficient concentration ofantibodies in the reaction mixture, such that the efficiency of theseparation is not limited by lack of antibody (i.e., using a saturatingamount of antibody). The appropriate concentration can also bedetermined by titration. The medium in which the cells are separatedwill be any medium that maintains the viability of the cells. Apreferred medium is phosphate buffered saline containing from 0.1% to0.5% BSA. Various media are commercially available and may be usedaccording to the nature of the cells, including Dulbecco's ModifiedEagle Medium, Hank's Basic Salt Solution, Dulbecco's phosphate bufferedsaline, RPMI, Iscove's medium, PBS with 5 mM EDTA, etc., optionallysupplemented with fetal calf serum, BSA, HSA, etc.

The staining intensity of cells can be monitored by flow cytometry,where lasers detect the quantitative levels of fluorochrome (which isproportional to the amount of cell surface antigen bound by theantibodies). Flow cytometry, or fluorescent activated cell sorting(FACS), can also be used to separate cell populations based on theintensity of antibody staining, as well as other parameters such as cellsize and light scatter. Although the absolute level of staining maydiffer with a particular fluorochrome and antibody preparation, the datacan be normalized to a control.

The labeled cells are then separated as to the expression of designatedmarker (e.g., CD4, CD25, etc.). The separated cells may be collected inany appropriate medium that maintains the viability of the cells,usually having a cushion of serum at the bottom of the collection tube.Various media are commercially available and may be used according tothe nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove'smedium, etc., frequently supplemented with fetal calf serum.

Cell populations highly enriched for a desired characteristic (e.g.,CD4⁺ T cells, CD4⁺CD25⁺ regulatory T cells, etc.) are achieved in thismanner. The desired population will be at or about 70% or more of thecell composition, and usually at or about 90% or more of the cellcomposition, and may be as much as about 95% or more of the cellpopulation. The enriched cell population may be used immediately. Cellscan also be frozen, although it is preferable to freeze cells prior tothe separation procedure. Alternatively, cells may be frozen at liquidnitrogen temperatures and stored for long periods of time, being thawedand capable of being reused. The cells will usually be stored in DMSOand/or FCS, in combination with medium, glucose, etc. Once thawed, thecells may be expanded by use of growth factors, antigen, stimulation,antigen presenting cells (e.g., dendritic cells), etc. for proliferationand differentiation.

Once the PBMCs or isolated T cells have undergone any necessarypre-treatment, the cells are treated with STRO-1⁺ cells and/or solublefactors derived therefrom. By “treated” herein is meant that the cellsare incubated in a suitable nutrient medium with STRO-1⁺ cells and/orsoluble factors derived therefrom for a time period sufficient toproduce regulatory T cells having the capacity to inhibit immuneresponses mediated by effector T cells. In some embodiments, the firstculture is diluted with about an equal volume of nutrient medium. Inother aspects, a first cell culture is divided into two or more portionsthat are then diluted with nutrient medium. The advantage of culturedivision is that the cell clusters formed in the first culture(thousands of cells) are mechanically disrupted and form smaller cellclusters (tens to hundreds of cells) during division of the firstculture. These small clusters are then able to grow into larger clustersduring the next growth period. A cell culture produced in this fashionmay be subcultured two or more times using a similar method.

A cell population may be grown in vitro under various cultureconditions. Culture medium may be liquid or semi-solid (e.g., containingagar, methylcellulose, etc.) The cell population may be convenientlysuspended in any appropriate nutrient medium, including but not limitedto Iscove's modified Dulbecco's medium, or RPMI-1640, normallysupplemented with fetal calf serum (about 5-10%), L-glutamine, andantibiotics (e.g., penicillin and streptomycin).

The cell culture may contain growth factors to which the cells areresponsive. Growth factors, as defined herein, are molecules capable ofpromoting survival, growth and/or differentiation of cells, either inculture or in the intact tissue, through specific effects on atransmembrane receptor. Growth factors include polypeptides andnon-polypeptide factors. Specific growth factors that may be used inculturing the subject cells include the interleukins (e.g., IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, etc.) and antigens (e.g., peptideantigens, protein antigens such as alloantigens) preferably incombination with antigen presenting cells, lectins, non-specific stimuli(e.g., Con A; LPS; etc.). The culture may also contain antibodies (e.g.anti-CD3), or specific ligands (in the Ruin of purified ligand, Fcfusion proteins, or other recombinant tagged forms like leucine zipperforms) for cell surface receptors that may stimulate regulatory T cellactivity. For example, mAb or ligands that bind TNFR or otherco-stimulatory molecules on regulatory T cells and could stimulate andincrease regulatory T cell activity.

The T cell population may be co-cultured with immature or maturedendritic cells, as well as other antigen presenting cells (e.g.,monocytes, B cells, macrophages, etc.) prior to, during, or aftertreatment STRO-1⁺ cells and/or soluble factors derived therefrom.

In some aspects, the present methods are useful for ex vivo generationof regulatory T cells for transplantation into a patient or developmentof in vitro models and assays for regulatory T cell function. Theregulatory T cell cultures serve as a valuable source of novelregulatory factors and pharmaceuticals.

Methods for adoptive transfer of T cells are well described in the art,for example, see US Patent Applications 2006/0115899, 2005/0196386,2003/0049696, 2006/0292164, and 2007/0172947 (the contents of which arehereby incorporated by reference). Therefore, a skilled practitionerwould easily be able to transplant or reintroduce the treated T cellspopulations obtained by the methods of the present disclosure into apatient in need thereof. Transplanted T cells may originate from a Tcell-containing sample obtained from the patient himself or from anotherdonor not receiving treatment. This is generally done as is known in theart and usually comprises injecting, or other methods of introducing,the treated cells back into the patient via intravenous administration.For example, the cells may be placed in a 50 ml Fenwall infusion bag byinjection using sterile syringes or other sterile transfer mechanisms.The cells can then be immediately infused via IV administration over aperiod of time, into a free flow IV line into the patient. In someaspects, additional reagents such as buffers or salts may be added aswell.

Another aspect of the disclosure provides methods for treatingautoimmune-related disorders by conjoint administration of treated Tcells obtained by a method of the present disclosure and at least oneadditional active agent. In some embodiments, the additional activeagent is a therapeutic agent used to treat or prevent an autoimmunedisease. Active agents of the invention may include, but are not limitedto beta-interferons, corticosteroids, non-steroid anti-inflammatorydrugs, tumor necrosis blockers, antimalarial drugs, cyclosporines, tumornecrosis alpha inhibitors, immunosuppressants, immunomodulators,cytokines, anti-graft-rejection therapeutics, vitamin D3, Dexamethasone,antibody therapeutics, and T cell epitopes (e.g., ToleroTrans TransplantRejection Therapy by Circassia, etc.). Cytokines suitable for conjointadministration may include, but are not limited to IL-2, IL-4, IL-7,IL-10, TGF-β, IL-15 and/or IL-17. In some embodiments the additionalactive agent may be a cell population comprising other cell types thanregulatory T cells. For example, the treated T cells may be conjointlyadministered to a patient in need thereof with one or more antigenpresenting cell types, such as monocytes or dendritic cells. In someaspects, these antigen presenting cells may be activated monocytes ordendritic cells.

After transplanting the cells into the patient, the effect of thetreatment may be evaluated, if desired. One of skill in the art wouldrecognize there are many methods of evaluating immunologicalmanifestations of an autoimmune disease (e.g., quantification of totalantibody titers or of specific immunoglobulins, renal function tests,tissue damage evaluation, etc.). Tests of T cells function such as Tcell numbers, phenotype, activation state and ability to respond toantigens and/or mitogens also may be done.

One aspect of the disclosure provides methods for treating or preventinga disorder caused by excessive T cell activation, such as an autoimmunedisorder or condition, in a patient by administering to a patient inneed thereof an amount of STRO-1⁺ cells and/or soluble factors derivedtherefrom effective to suppress T-cell activation in the patient.

The examples of the disclosure demonstrate that administration ofSTRO-1⁺ cells and/or soluble factors derived therefrom to a mouse modelresulted in suppression of effector T cell activity.

In one embodiment the present disclosure provides methods ofadministering STRO-1⁺ cells and/or soluble factors derived therefrom topromote regulatory T cell-mediated suppression of autoimmune disordersor conditions.

While the method of the invention can be used to treat patientsafflicted with an autoimmune disorder, in some embodiments, the methodsare also applied to patients who do not have, but are at risk ofdeveloping an autoimmune response.

The present disclosure provides methods for treating autoimmune-relateddisorders by conjoint administration of STRO-1⁺ cells and/or solublefactors derived therefrom and at least one additional active agent.Active agents of the invention may include, but are not limited tobeta-interferons, corticosteroids, non-steroid anti-inflammatory drugs,tumor necrosis blockers, antimalarial drugs, cyclosporines, tumornecrosis alpha inhibitors, immunosuppressants, immunomodulators,cytokines, anti-graft-rejection therapeutics, cell-based therapeutics,vitamin D3, dexamethasone and antibody therapeutics. Cytokines suitablefor conjoint administration may include, but are not limited to IL-2,IL-4, IL-10, TGF-β, IL-15 and/or IL-17. In some embodiments, theadditional active agent is a therapeutic agent used to treat or preventan autoimmune disease.

The pathogenesis of a number of autoimmune diseases is believed to becaused by autoimmune T cell responses to self-antigens present in theorganism. For example, autoreactive T cells have been implicated in thepathogenesis of: type I diabetes, multiple sclerosis, rheumatoidarthritis, psoriatic arthritis, autoimmune myocarditis, pemphigus,celiac disease, myasthenia gravis, Hashimoto's thyroiditis, Graves'disease, Addison's disease, autoimmune hepatitis, chronic Lymearthritis, familial dilated cardiomyopathy, juvenile dermatomyositis,polychondritis, Sjogren's syndrome, psoriasis, juvenile idiopathicarthritis, inflammatory bowel disease, systemic lupus erythematosus, andgraft-versus-host disease.

As used herein, the term “soluble factors” shall be taken to mean anymolecule, e.g., protein, peptide, glycoprotein, glycopeptide,lipoprotein, lipopeptide, carbohydrate, etc. produced by STRO-1⁺ cellsand/or progeny thereof that are water soluble. Such soluble factors maybe intracellular and/or secreted by a cell. Such soluble factors may bea complex mixture (e.g., supernatant) and/or a fraction thereof and/ormay be a purified factor. In one embodiment of the present inventionsoluble factors are or are contained within supernatant. Accordingly,any embodiment herein directed to administration of one or more solublefactors shall be taken to apply mutatis mutandis to the administrationof supernatant.

The methods of the invention may involve administration of population ofcells enriched for STRO-1⁺ cells and/or progeny cells thereof alone,and/or soluble factors derived therefrom. The methods of the inventionmay also involve administration of progeny cells alone, or solublefactors derived from the progeny cells. The methods of the invention mayalso involve administration of a mixed population of STRO-1^(bri) cellsand progeny cells thereof, or soluble factors from a mixed culture ofSTRO-1^(bri) cells and progeny cells thereof.

It is further contemplated that only a single treatment with the STRO-1⁺cells and/or progeny cells thereof and/or soluble factors derivedtherefrom of the present invention may be required, eliminating the needfor chronic immunosuppressive drug therapy. Alternatively, multipleadministrations of STRO-1⁺ cells and/or progeny cells thereof and/orsoluble factors derived therefrom may be employed.

The dosage of the STRO-1⁺ cells and/or progeny cells thereof and/orsoluble factors derived therefrom varies within wide limits and will, ofcourse be fitted to the individual requirements in each particular case.In general, in the case of parenteral administration, it is customary toadminister from about 0.01 to about 5 million cells per kilogram ofrecipient body weight. The number of cells used will depend on theweight and condition of the recipient, the number of or frequency ofadministrations, and other variables known to those of skill in the art.

Exemplary dosages of the cells include between 0.1×10⁶ to 5×10⁶ STRO-1⁺cells and/or progeny thereof. For example, the method comprisesadministering between 0.3×10⁶ to 2×10⁶ STRO-1⁺ cells and/or progenythereof.

One form of the method involves administering a low dose of STRO-1⁺cells and/or progeny thereof. Such a low dose is, for example, between0.1×10⁵ and 0.5×10⁶ STRO-1⁺ cells and/or progeny thereof, such as about0.3×10⁶ STRO-1⁺ cells and/or progeny thereof.

The cells can be suspended in an appropriate diluent, at a concentrationof from about 0.01 to about 5×10⁶ cells/ml. Suitable excipients forinjection solutions are those that are biologically and physiologicallycompatible with the cells and with the recipient, such as bufferedsaline solution or other suitable excipients. The composition foradministration is preferably formulated, produced and stored accordingto standard methods complying with proper sterility and stability.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

STRO-1⁺ Cells or Progeny Cells, and Supernatant or One or More SolubleFactors Derived Therefrom

STRO-1⁺ cells are cells found in bone marrow, blood, dental pulp cells,adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart,retina, brain, hair follicles, intestine, lung, lymph node, thymus,bone, ligament, tendon, skeletal muscle, dermis, and periosteum; and arecapable of differentiating into germ lines such as mesoderm and/orendoderm and/or ectoderm.

In one embodiment, the STRO-1⁺ cells are multipotential cells which arecapable of differentiating into a large number of cell types including,but not limited to, adipose, osseous, cartilaginous, elastic, muscular,and fibrous connective tissues. The specific lineage-commitment anddifferentiation pathway which these cells enter depends upon variousinfluences from mechanical influences and/or endogenous bioactivefactors, such as growth factors, cytokines, and/or localmicroenvironmental conditions established by host tissues. STRO-1⁺multipotential cells are thus non-hematopoietic progenitor cells whichdivide to yield daughter cells that are either stem cells or areprecursor cells which in time will irreversibly differentiate to yield aphenotypic cell.

In one example, the STRO-1⁺ cells are enriched from a sample obtainedfrom a subject, e.g., a subject to be treated or a related subject or anunrelated subject (whether of the same species or different). The terms‘enriched’, ‘enrichment’ or variations thereof are used herein todescribe a population of cells in which the proportion of one particularcell type or the proportion of a number of particular cell types isincreased when compared with an untreated population of the cells (e.g.,cells in their native environment).

In one example, the cells used in the present disclosure express one ormore markers individually or collectively selected from the groupconsisting of TNAP⁺, VCAM-1⁺, THY-1⁺, STRO-4⁺ (HSP-90β), STRO-2⁺, CD45⁺,CD146⁺, 3G5⁺ or any combination thereof.

By “individually” is meant that the disclosure encompasses the recitedmarkers or groups of markers separately, and that, notwithstanding thatindividual markers or groups of markers may not be separately listedherein the accompanying claims may define such marker or groups ofmarkers separately and divisibly from each other.

By “collectively” is meant that the disclosure encompasses any number orcombination of the recited markers or groups of peptides, and that,notwithstanding that such numbers or combinations of markers or groupsof markers may not be specifically listed herein the accompanying claimsmay define such combinations or sub-combinations separately anddivisibly from any other combination of markers or groups of markers.

In one example, the STRO-1⁺ cells are STRO-1^(bright) (syn.STRO-1^(bri)). In one example, the Stro-1^(bri) cells are preferentiallyenriched relative to STRO-1^(dim) or STRO-1^(intermediate) cells.

In one example, the STRO-1^(bright) cells are additionally one or moreof TNAP⁺, VCAM-1⁺, THY-1⁺, STRO-4⁺ (HSP-90β), STRO-2⁺ and/or CD146⁺.

In one example, the mesenchymal precursor cells are perivascularmesenchymal precursor cells as defined in WO 2004/85630.

A cell that is referred to as being “positive” for a given marker it mayexpress either a low (lo or dim) or a high (bright, bri) level of thatmarker depending on the degree to which the marker is present on thecell surface, where the terms relate to intensity of fluorescence orother marker used in the sorting process of the cells. The distinctionof lo (or dim or dull) and bri will be understood in the context of themarker used on a particular cell population being sorted. A cell that isreferred to as being “negative” for a given marker is not necessarilycompletely absent from that cell. This term means that the marker isexpressed at a relatively very low level by that cell, and that itgenerates a very low signal when detectably labeled or is undetectableabove background levels, e.g., levels detected suing an isotype controlantibody.

The term “bright”, when used herein, refers to a marker on a cellsurface that generates a relatively high signal when detectably labeled.Whilst not wishing to be limited by theory, it is proposed that “bright”cells express more of the target marker protein (for example the antigenrecognized by STRO-1) than other cells in the sample. For instance,STRO-1^(bri) cells produce a greater fluorescent signal, when labeledwith a FITC-conjugated STRO-1 antibody as determined by fluorescenceactivated cell sorting (FACS) analysis, than non-bright cells(STRO-1^(dull/dim)). In one example, “bright” cells constitute at leastabout 0.1% of the most brightly labeled bone marrow mononuclear cellscontained in the starting sample. In other examples, “bright” cellsconstitute at least about 0.1%, at least about 0.5%, at least about 1%,at least about 1.5%, or at least about 2%, of the most brightly labeledbone marrow mononuclear cells contained in the starting sample. In anexample, STRO-1^(bright) cells have 2 log magnitude higher expression ofSTRO-1 surface expression relative to “background”, namely cells thatare STRO-1⁻. By comparison, STRO-1^(dim) and/or STRO-1^(intermediate)cells have less than 2 log magnitude higher expression of STRO-1 surfaceexpression, typically about 1 log or less than “background”.

As used herein the term “TNAP” is intended to encompass all isoforms oftissue non-specific alkaline phosphatase. For example, the termencompasses the liver isoform (LAP), the bone isoform (BAP) and thekidney isoform (KAP). In an example, the TNAP is BAP. In an example,TNAP as used herein refers to a molecule which can bind the STRO-3antibody produced by the hybridoma cell line deposited with ATCC on 19Dec. 2005 under the provisions of the Budapest Treaty under depositaccession number PTA-7282.

Furthermore, in an example, the STRO-1⁺ cells are capable of giving riseto clonogenic CFU-F.

In one example, a significant proportion of the STRO-1⁺ multipotentialcells are capable of differentiation into at least two different germlines. Non-limiting examples of the lineages to which the multipotentialcells may be committed include bone precursor cells; hepatocyteprogenitors, which are multipotent for bile duct epithelial cells andhepatocytes; neural restricted cells, which can generate glial cellprecursors that progress to oligodendrocytes and astrocytes; neuronalprecursors that progress to neurons; precursors for cardiac muscle andcardiomyocytes, glucose-responsive insulin secreting pancreatic betacell lines. Other lineages include, but are not limited to,odontoblasts, dentin-producing cells and chondrocytes, and precursorcells of the following: retinal pigment epithelial cells, fibroblasts,skin cells such as keratinocytes, dendritic cells, hair follicle cells,renal duct epithelial cells, smooth and skeletal muscle cells,testicular progenitors, vascular endothelial cells, tendon, ligament,cartilage, adipocyte, fibroblast, marrow stroma, cardiac muscle, smoothmuscle, skeletal muscle, pericyte, vascular, epithelial, glial,neuronal, astrocyte and oligodendrocyte cells.

In another example, the STRO-1⁺ cells are not capable of giving rise,upon culturing, to hematopoietic cells.

In one example, the cells are taken from the subject to be treated,cultured in vitro using standard techniques and used to obtainsupernatant or soluble factors or expanded cells for administration tothe subject as an autologous or allogeneic composition. In analternative example, cells of one or more of the established human celllines are used. In another useful example of the disclosure, cells of anon-human animal (or if the patient is not a human, from anotherspecies) are used.

The present disclosure also contemplates use of supernatant or solublefactors obtained or derived from STRO-1⁺ cells and/or progeny cellsthereof (the latter also being referred to as expanded cells) which areproduced from in vitro culture. Expanded cells of the disclosure may ahave a wide variety of phenotypes depending on the culture conditions(including the number and/or type of stimulatory factors in the culturemedium), the number of passages and the like. In certain examples, theprogeny cells are obtained after about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, or about 10 passages from theparental population. However, the progeny cells may be obtained afterany number of passages from the parental population.

The progeny cells may be obtained by culturing in any suitable medium.The term “medium”, as used in reference to a cell culture, includes thecomponents of the environment surrounding the cells. Media may be solid,liquid, gaseous or a mixture of phases and materials. Media includeliquid growth media as well as liquid media that do not sustain cellgrowth. Media also include gelatinous media such as agar, agarose,gelatin and collagen matrices. Exemplary gaseous media include thegaseous phase that cells growing on a petri dish or other solid orsemisolid support are exposed to. The term “medium” also refers tomaterial that is intended for use in a cell culture, even if it has notyet been contacted with cells. In other words, a nutrient rich liquidprepared for bacterial culture is a medium. A powder mixture that whenmixed with water or other liquid becomes suitable for cell culture maybe termed a “powdered medium”.

In an example, progeny cells useful for the methods of the disclosureare obtained by isolating TNAP⁺ STRO-1⁺ cells from bone marrow usingmagnetic beads labeled with the STRO-3 antibody, and then cultureexpanding the isolated cells (see Gronthos et al. Blood 85: 929-940,1995 for an example of suitable culturing conditions).

In one example, such expanded cells (progeny) (for example, at leastafter 5 passages) can be TNAP⁻, CC9⁺, HLA class I⁺, HLA class II⁻,CD14⁻, CD19⁻, CD3⁻, CD11a⁻c⁻, CD31⁻, CD86⁻, CD34⁻ and/or CD80⁻. However,it is possible that under different culturing conditions to thosedescribed herein that the expression of different markers may vary.Also, whilst cells of these phenotypes may predominate in the expendedcell population it does not mean that there is a minor proportion of thecells do not have this phenotype(s) (for example, a small percentage ofthe expanded cells may be CC9⁻). In one example, expanded cells stillhave the capacity to differentiate into different cell types.

In one example, an expended cell population used to obtain supernatantor soluble factors, or cells per se, comprises cells wherein at least25%, such as at least 50%, of the cells are CC9⁺.

In another example, an expanded cell population used to obtainsupernatant or soluble factors, or cells per se, comprises cells whereinat least 40%, such as at least 45%, of the cells are STRO-1⁺.

In a further example, the expanded cells may express one or more markerscollectively or individually selected from the group consisting ofLFA-3, THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, 3G5,CD49a/CD49b/CD29, CD49c/CD29, CD49d/CD29, CD 90, CD29, CD18, CD61,integrin beta 6-19, thrombomodulin, CD10, CD13, SCF, PDGF-R, EGF-R,IGF1-R, NGF-R, FGF-R, Leptin-R (STRO-2=Leptin-R), RANKL, STRO-1^(bright)and CD146 or any combination of these markers.

In one example, the progeny cells are Multipotential Expanded STRO-1⁺Multipotential cells Progeny (MEMPs) as defined and/or described in WO2006/032092. Methods for preparing enriched populations of STRO-1⁺multipotential cells from which progeny may be derived are described inWO 01/04268 and WO 2004/085630. In an in vitro context STRO-1⁺multipotential cells will rarely be present as an absolutely purepreparation and will generally be present with other cells that aretissue specific committed cells (TSCCs). WO 01/04268 refers toharvesting such cells from bone marrow at purity levels of about 0.1% to90%. The population comprising MPCs from which progeny are derived maybe directly harvested from a tissue source, or alternatively it may be apopulation that has already been expanded ex vivo.

For example, the progeny may be obtained from a harvested, unexpanded,population of substantially purified STRO-1⁺ multipotential cells,comprising at least about 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80 or95% of total cells of the population in which they are present. Thislevel may be achieved, for example, by selecting for cells that arepositive for at least one marker individually or collectively selectedfrom the group consisting of TNAP, STRO-1^(bright), 3G5⁺, VCAM-1, THY-1,CD146 and STRO-2.

MEMPS can be distinguished from freshly harvested STRO-1⁺ multipotentialcells in that they are positive for the marker STRO-1^(bri) and negativefor the marker Alkaline phosphatase (ALP). In contrast, freshly isolatedSTRO-1⁺ multipotential cells are positive for both STRO-1^(bri) and ALP.In an example of the present disclosure, at least 15%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or 95% of the administered cells have thephenotype STRO-1^(bri), ALP⁻. In one example the MEMPS are positive forone or more of the markers Ki67, CD44 and/or CD49c/CD29, VLA-3, α3β1. Inyet a further example the MEMPs do not exhibit TERT activity and/or arenegative for the marker CD18.

The STRO-1⁺ cell starting population may be derived from any one or moretissue types set out in WO 01/04268 or WO 2004/085630, namely bonemarrow, dental pulp cells, adipose tissue and skin, or perhaps morebroadly from adipose tissue, teeth, dental pulp, skin, liver, kidney,heart, retina, brain, hair follicles, intestine, lung, spleen, lymphnode, thymus, pancreas, bone, ligament, bone marrow, tendon and skeletalmuscle.

It will be understood that in performing the present disclosure,separation of cells carrying any given cell surface marker can beeffected by a number of different methods, however, some methods relyupon binding a binding agent (e.g., an antibody or antigen bindingfragment thereof) to the marker concerned followed by a separation ofthose that exhibit binding, being either high level binding, or lowlevel binding or no binding. The most convenient binding agents areantibodies or antibody-based molecules, such as monoclonal antibodies orbased on monoclonal antibodies because of the specificity of theselatter agents. Antibodies can be used for both steps, however otheragents might also be used, thus ligands for these markers may also beemployed to enrich for cells carrying them, or lacking them.

The antibodies or ligands may be attached to a solid support to allowfor a crude separation. The separation techniques preferably maximizethe retention of viability of the fraction to be collected. Varioustechniques of different efficacy may be employed to obtain relativelycrude separations. The particular technique employed will depend uponefficiency of separation, associated cytotoxicity, ease and speed ofperformance, and necessity for sophisticated equipment and/or technicalskill. Procedures for separation may include, but are not limited to,magnetic separation, using antibody-coated magnetic beads, affinitychromatography and “panning” with antibody attached to a solid matrix.Techniques providing accurate separation include but are not limited toFACS. Methods for performing FACS will be apparent to the skilledartisan.

Antibodies against each of the markers described herein are commerciallyavailable (e.g., monoclonal antibodies against STRO-1 are commerciallyavailable from R&D Systems, USA), available from ATCC or otherdepositary organization and/or can be produced using art recognizedtechniques.

The method for isolating STRO-1⁺ cells, for example, comprises a firststep being a solid phase sorting step utilizing for example magneticactivated cell sorting (MACS) recognizing high level expression ofSTRO-1. A second sorting step can then follow, should that be desired,to result in a higher level of precursor cell expression as described inpatent specification WO 01/14268. This second sorting step might involvethe use of two or more markers.

The method obtaining STRO-1⁺ cells might also include the harvesting ofa source of the cells before the first enrichment step using knowntechniques. Thus the tissue will be surgically removed. Cells comprisingthe source tissue will then be separated into a so called single cellssuspension. This separation may be achieved by physical and or enzymaticmeans.

Once a suitable STRO-1⁺ cell population has been obtained, it may becultured or expanded by any suitable means to obtain MEMPs.

In one example, the cells are taken from the subject to be treated,cultured in vitro using standard techniques and used to obtainsupernatant or soluble factors or expanded cells for administration tothe subject as an autologous or allogeneic composition. In analternative example, cells of one or more of the established human celllines are used to obtain the supernatant or soluble factors. In anotheruseful example of the disclosure, cells of a non-human animal (or if thepatient is not a human, from another species) are used to obtainsupernatant or soluble factors.

The disclosure can be practiced using cells from any non-human animalspecies, including but not limited to non-human primate cells, ungulate,canine, feline, lagomorph, rodent, avian, and fish cells. Primate cellswith which the disclosure may be performed include but are not limitedto cells of chimpanzees, baboons, cynomolgus monkeys, and any other Newor Old World monkeys. Ungulate cells with which the disclosure may beperformed include but are not limited to cells of bovines, porcines,ovines, caprines, equines, buffalo and bison. Rodent cells with whichthe disclosure may be performed include but are not limited to mouse,rat, guinea pig, hamster and gerbil cells. Examples of lagomorph specieswith which the disclosure may be performed include domesticated rabbits,jack rabbits, hares, cottontails, snowshoe rabbits, and pikas. Chickens(Gallus gallus) are an example of an avian species with which thedisclosure may be performed.

Cells useful for the methods of the disclosure may be stored before use,or before obtaining the supernatant or soluble factors. Methods andprotocols for preserving and storing of eukaryotic cells, and inparticular mammalian cells, are known in the art (cf., for example,Pollard, J. W. and Walker, J. M. (1997) Basic Cell Culture Protocols,Second Edition, Humana Press, Totowa, N.J.; Freshney, R. I. (2000)Culture of Animal Cells, Fourth Edition, Wiley-Liss, Hoboken, N.J.). Anymethod maintaining the biological activity of the isolated stem cellssuch as mesenchymal stem/progenitor cells, or progeny thereof, may beutilized in connection with the present disclosure. In one example, thecells are maintained and stored by using cryo-preservation.

Genetically-Modified Cells

In one embodiment, the STRO-1⁺ cells and/or progeny cells thereof aregenetically modified, e.g., to express and/or secrete a protein ofinterest, e.g., a protein providing a therapeutic and/or prophylacticbenefit, e.g., insulin, glucagon, somatostatin, trypsinogen,chymotrypsinogen, elastase, carboxypeptidase, pancreatic lipase oramylase or a polypeptide associated with or causative of enhancedangiogenesis or a polypeptide associated with differentiation of a cellinto a pancreatic cell or a vascular cell.

Methods for genetically modifying a cell will be apparent to the skilledartisan. For example, a nucleic acid that is to be expressed in a cellis operably-linked to a promoter for inducing expression in the cell.For example, the nucleic acid is linked to a promoter operable in avariety of cells of a subject, such as, for example, a viral promoter,e.g., a CMV promoter (e.g., a CMV-IE promoter) or a SV-40 promoter.Additional suitable promoters are known in the art and shall be taken toapply mutatis mutandis to the present embodiment of the invention.

Preferably, the nucleic acid is provided in the form of an expressionconstruct. As used herein, the term “expression construct” refers to anucleic acid that has the ability to confer expression on a nucleic acid(e.g. a reporter gene and/or a counter-selectable reporter gene) towhich it is operably connected, in a cell. Within the context of thepresent invention, it is to be understood that an expression constructmay comprise or be a plasmid, bacteriophage, phagemid, cosmid, virussub-genomic or genomic fragment, or other nucleic acid capable ofmaintaining and/or replicating heterologous DNA in an expressibleformat.

Methods for the construction of a suitable expression construct forperformance of the invention will be apparent to the skilled artisan andare described, for example, in Ausubel et al (In: Current Protocols inMolecular Biology. Wiley Interscience, ISBN 047 150338, 1987) orSambrook et al (In: Molecular Cloning: Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).For example, each of the components of the expression construct isamplified from a suitable template nucleic acid using, for example, PCRand subsequently cloned into a suitable expression construct, such asfor example, a plasmid or a phagemid.

Vectors suitable for such an expression construct are known in the artand/or described herein. For example, an expression vector suitable forthe method of the present invention in a mammalian cell is, for example,a vector of the pcDNA vector suite supplied by Invitrogen, a vector ofthe pCI vector suite (Promega), a vector of the pCMV vector suite(Clontech), a pM vector (Clontech), a pSI vector (Pro ega), a VP 16vector (Clontech) or a vector of the pcDNA vector suite (Invitrogen).

The skilled artisan will be aware of additional vectors and sources ofsuch vectors, such as, for example, Invitrogen Corporation, Clontech orPromega.

Means for introducing the isolated nucleic acid molecule or a geneconstruct comprising same into a cell for expression are known to thoseskilled in the art. The technique used for a given organism depends onthe known successful techniques. Means for introducing recombinant DNAinto cells include microinjection, transfection mediated byDEAE-dextran, transfection mediated by liposomes such as by usinglipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA),PEG-mediated DNA uptake, electroporation and microparticle bombardmentsuch as by using DNA-coated tungsten or gold particles (Agracetus Inc.,WI, USA) amongst others.

Alternatively, an expression construct of the invention is a viralvector. Suitable viral vectors are known in the art and commerciallyavailable. Conventional viral-based systems for the delivery of anucleic acid and integration of that nucleic acid into a host cellgenome include, for example, a retroviral vector, a lentiviral vector oran adeno-associated viral vector. Alternatively, an adenoviral vector isuseful for introducing a nucleic acid that remains episomal into a hostcell. Viral vectors are an efficient and versatile method of genetransfer in target cells and tissues. Additionally, high transductionefficiencies have been observed in many different cell types and targettissues.

For example, a retroviral vector generally comprises cis-acting longterminal repeats (LTRs) with packaging capacity for up to 6-10 kb offoreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of a vector, which is then used to integratethe expression construct into the target cell to provide long termexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simianimmunodeficiency virus (SrV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J Virol. 56:2731-2739(1992); Johann et al, J. Virol. 65:1635-1640 (1992); Sommerfelt et al,Virol. 76:58-59 (1990); Wilson et al, J. Virol. 63:274-2318 (1989);Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700; Miller andRosman BioTechniques 7:980-990, 1989; Miller, A. D. Human Gene Therapy7:5-14, 1990; Scarpa et al Virology 75:849-852, 1991; Burns et al. Proc.Natl. Acad. Sci USA 90:8033-8037, 1993).

Various adeno-associated virus (AAV) vector systems have also beendeveloped for nucleic acid delivery. AAV vectors can be readilyconstructed using techniques known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 andWO 93/03769; Lebkowski et al. Molec. Cell. Biol. 5:3988-3996, 1988;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor LaboratoryPress);Carter Current Opinion in Biotechnology 5:533-539, 1992;Muzyczka. Current Topics in Microbial, and Immunol. 158:97-129, 1992;Kotin, Human Gene Therapy 5:793-801, 1994; Shelling and Smith GeneTherapy 7:165-169, 1994; and Zhou et al. J Exp. Med. 179:1867-1875,1994.

Additional viral vectors useful for delivering an expression constructof the invention include, for example, those derived from the pox familyof viruses, such as vaccinia virus and avian poxvirus or an alphavirusor a conjugate virus vector (e.g. that described in Fisher-Hoch et al.,Proc. Natl Acad. Sci. USA 56:317-321, 1989).

Assaying Therapeutic/Prophylactic Potential of Cells and Soluble Factors

Methods for determining the ability of soluble factors derived fromSTRO-1^(bright) cells to suppress T-cell activation will be apparent tothe skilled artisan.

For example, suitable in vitro tests for determining immunosuppressiveactivity of the soluble factors are described in Examples 5 and 8herein.

In another example, efficacy of cells and/or soluble factors describedherein is assessed in an in vivo model of experimental inflammatoryencephalomyelitis (EAE) as described in Example 6 herein.

It will be apparent to the skilled artisan from the foregoing that thepresent disclosure also provides a method for identifying or isolating asoluble factor for suppressing T cell activation, the method comprising:

(i) administering a a soluble factor to a test subject suffering fromEAE and assessing progression of EAE in the subject;

(ii) comparing level of EAE in the subject at (i) to the level EAE in acontrol subject suffering from EAE to which the soluble factor has notbeen administered, wherein reduced EAE in the test subject compared tothe control subject indicates that the soluble factor treats, preventsor delays EAE.

Cellular Compositions

In one embodiment of the present invention STRO-1⁺ cells and/or progenycells thereof are administered in the form of a composition. Preferably,such a composition comprises a pharmaceutically acceptable carrierand/or excipient.

The terms “carrier” and “excipient” refer to compositions of matter thatare conventionally used in the art to facilitate the storage,administration, and/or the biological activity of an active compound(see, e.g., Remington's Pharmaceutical Sciences, 16th Ed., MacPublishing Company (1980). A carrier may also reduce any undesirableside effects of the active compound. A suitable carrier is, for example,stable, e.g., incapable of reacting with other ingredients in thecarrier. In one example, the carrier does not produce significant localor systemic adverse effect in recipients at the dosages andconcentrations employed for treatment.

Suitable carriers for this invention include those conventionally used,e.g., water, saline, aqueous dextrose, lactose, Ringer's solution, abuffered solution, hyaluronan and glycols are preferred liquid carriers,particularly (when isotonic) for solutions. Suitable pharmaceuticalcarriers and excipients include starch, cellulose, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesiumstearate, sodium stearate, glycerol monostearate, sodium chloride,glycerol, propylene glycol, water, ethanol, and the like.

In another example, a carrier is a media composition, e.g., in which acell is grown or suspended. Preferably, such a media composition doesnot induce any adverse effects in a subject to whom it is administered.

Preferred carriers and excipients do not adversely affect the viabilityof a cell and/or the ability of a cell to reduce, prevent or delaypancreatic dysfunction.

In one example, the carrier or excipient provides a buffering activityto maintain the cells and/or soluble factors at a suitable pH to therebyexert a biological activity, e.g., the carrier or excipient is phosphatebuffered saline (PBS). PBS represents an attractive carrier or excipientbecause it interacts with cells and factors minimally and permits rapidrelease of the cells and factors, in such a case, the composition of theinvention may be produced as a liquid for direct application to theblood stream or into a tissue or a region surrounding or adjacent to atissue, e.g., by injection.

STRO-1⁺ cells and/or progeny cells thereof can also be incorporated orembedded within scaffolds that are recipient-compatible and whichdegrade into products that are not harmful to the recipient. Thesescaffolds provide support and protection for cells that are to betransplanted into the recipient subjects. Natural and/or syntheticbiodegradable scaffolds are examples of such scaffolds.

A variety of different scaffolds may be used successfully in thepractice of the invention. Preferred scaffolds include, but are notlimited to biological, degradable scaffolds. Natural biodegradablescaffolds include collagen, fibronectin, and laminin scaffolds. Suitablesynthetic material for a cell transplantation scaffold should be able tosupport extensive cell growth and cell function. Such scaffolds may alsobe resorbable. Suitable scaffolds include polyglycolic acid scaffolds,e.g., as described by Vacanti, et al. J. Ped. Surg. 23:3-9 1988; Cima,et al. Biotechnol. Bioeng. 38:145 1991; Vacanti, et al. Plast. Reconstr.Surg. 88:753-9 1991; or synthetic polymers such as polyanhydrides,polyorthoesters, and polylactic acid.

In another example, the cells may be administered in a gel scaffold(such as Gelfoam from Upjohn Company).

The cellular compositions useful for the present invention may beadministered alone or as admixtures with other cells. Cells that may beadministered in conjunction with the compositions of the presentinvention include, but are not limited to, other multipotent orpluripotent cells or stem cells, or bone marrow cells. The cells ofdifferent types may be admixed with a composition of the inventionimmediately or shortly prior to administration, or they may beco-cultured together for a period of time prior to administration.

Preferably, the composition comprises an effective amount or atherapeutically or prophylactically effective amount of cells. Forexample, the composition comprises about 1×10⁵ STRO-1⁺ cells/kg to about1×10⁷ STRO-1⁺ cells/kg or about 1×10⁶ STRO-1⁺ cells/kg to about 5×10⁶STRO-1⁺ cells/kg. The exact amount of cells to be administered isdependent upon a variety of factors, including the age, weight, and sexof the patient, and the extent and severity of the pancreaticdysfunction.

In some embodiments, cells are contained within a chamber that does notpermit the cells to exit into a subject's circulation, however thatpermits factors secreted by the cells to enter the circulation. In thismanner soluble factors may be administered to a subject by permittingthe cells to secrete the factors into the subject's circulation. Such achamber may equally be implanted at a site in a subject to increaselocal levels of the soluble factors, e.g., implanted in or near atransplanted organ.

In some embodiments of the invention, it may not be necessary ordesirable to immunosuppress a patient prior to initiation of therapywith cellular compositions. Accordingly, transplantation withallogeneic, or even xenogeneic, Stro-1^(bri) cells or progeny thereofmay be tolerated in some instances.

However, in other instances it may be desirable or appropriate topharmacologically immunosuppress a patient prior to initiating celltherapy. This may be accomplished through the use of systemic or localimmunosuppressive agents, or it may be accomplished by delivering thecells in an encapsulated device. The cells may be encapsulated in acapsule that is peiineable to nutrients and oxygen required by the celland therapeutic factors the cell is yet impermeable to immune humoralfactors and cells. Preferably the encapsulant is hypoallergenic, iseasily and stably situated in a target tissue, and provides addedprotection to the implanted structure. These and other means forreducing or eliminating an immune response to the transplanted cells areknown in the art. As an alternative, the cells may be geneticallymodified to reduce their immunogenicity.

Compositions of Soluble Factors

In one embodiment of the present invention, STRO-1⁺ cell-derived and/orprogeny cell-derived supernatant or soluble factors are administered inthe form of a composition, e.g., comprising a suitable carrier and/orexcipient. Preferably, the carrier or excipient does not adverselyaffect the biological effect of the soluble factors or supernatant.

In one embodiment, the composition comprises a composition of matter tostabilize a soluble factor or a component of supernatant, e.g., aprotease inhibitor. Preferably, the protease inhibitor is not includedin an amount sufficient to have an adverse effect on a subject.

Compositions comprising STRO-1⁺ cell-derived and/or progeny cell-derivedsupernatant or soluble factors may be prepared as appropriate liquidsuspensions, e.g., in culture medium or in a stable carrier or a buffersolution, e.g., phosphate buffered saline. Suitable carriers aredescribed herein above. In another example, suspensions comprisingSTRO-1⁺ cell-derived and/or progeny cell-derived supernatant or solublefactors are oily suspensions for injection. Suitable lipophilic solventsor vehicles include fatty oils such as sesame oil; or synthetic fattyacid esters, such as ethyl oleate or triglycerides; or liposomes.Suspensions to be used for injection may also contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe compounds to allow for the preparation of highly concentratedsolutions.

Sterile injectable solutions can be prepared by incorporating thesupernatant or soluble factors in the required amount in an appropriatesolvent with one or a combination of ingredients described above, asrequired, followed by filtered sterilization.

Generally, dispersions are prepared by incorporating the supernatant orsoluble factors into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. In accordance with an alternative aspect of theinvention, the supernatant or soluble factors may be formulated with oneor more additional compounds that enhance its solubility.

Other exemplary can iers or excipients are described, for example, inHardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis ofTherapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: TheScience and Practice of Pharmacy, Lippincott, Williams, and Wilkins, NewYork, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms:Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.)(1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY;Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: DisperseSystems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) ExcipientToxicity and Safety, Marcel Dekker, Inc., New York, N.Y.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, liposome, or other orderedstructure. The carrier can be a solvent or dispersion medium containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol, orsodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, monostearatesalts and gelatin. Moreover, the soluble factors may be administered ina time release formulation, for example in a composition which includesa slow release polymer. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, polylactic acid and polylactic, polyglycoliccopolymers (PLG). Many methods for the preparation of such formulationsare patented or generally known to those skilled in the art.

The supernatant or soluble factors may be administered in combinationwith an appropriate matrix, for instance, to provide slow release of thesoluble factors.

Modes of Administration

The STRO-1⁺ cell-derived supernatant or soluble factors, STRO-1⁺ cellsor progeny thereof may be surgically implanted, injected, delivered(e.g., by way of a catheter or syringe), or otherwise administereddirectly or indirectly to the site in need of repair or augmentation,e.g., an organ or into the blood system of a subject.

Preferably, the STRO-1⁺ cell-derived supernatant or soluble factors,STRO-1⁺ cells or progeny thereof is delivered to the blood stream of asubject. For example, the Stro-1^(bri) cell-derived supernatant orsoluble factors, STRO-1⁺ cells or progeny thereof are deliveredparenterally. Exemplary routes of parenteral administration include, butare not limited to, intravenous, intramuscular, subcutaneous,intra-arterial, intraperitoneal, intraventricular,intracerebroventricular, intrathecal. Preferably, the STRO-1⁺cell-derived supernatant or soluble factors, STRO-1⁺ cells or progenythereof are delivered intra-arterially, into an aorta, into an atrium orventricle of the heart or into a blood vessel connected to a pancreas,e.g., an abdominal aorta, a superior mesenteric artery, apancreaticoduodenal artery or a splenic artery.

In the case of cell delivery to an atrium or ventricle of the heart, itis preferred that cells are administered to the left atrium or ventricleto avoid complications that may arise from rapid delivery of cells tothe lungs.

Preferably, the STRO-1⁺ cell-derived supernatant or soluble factors,STRO-1⁺ cells or progeny thereof are injected into the site of delivery,e.g., using a syringe or through a catheter or a central line.

Selecting an administration regimen for a therapeutic formulationdepends on several factors, including the serum or tissue turnover rateof the entity, the level of symptoms, and the immunogenicity of theentity. Preferably, an administration regimen maximizes the amount oftherapeutic compound delivered to the patient consistent with anacceptable level of side effects. Accordingly, the amount of formulationdelivered depends in part on the particular entity and the severity ofthe condition being treated.

In one embodiment, STRO-1⁺ cell-derived supernatant or soluble factors,STRO-1⁺ cells or progeny thereof are delivered as a single bolus dose.Alternatively, STRO-1⁺ cell-derived supernatant or soluble factors,STRO-1⁺ cells or progeny thereof are administered by continuousinfusion, or by doses at intervals of, e.g., one day, one week, or 1-7times per week. A preferred dose protocol is one involving the maximaldose or dose frequency that avoids significant undesirable side effects.A total weekly dose depends on the type and activity of the compoundbeing used. Determination of the appropriate dose is made by aclinician, e.g., using parameters or factors known or suspected in theart to affect treatment or predicted to affect treatment. Generally, thedose begins with an amount somewhat less than the optimum dose and isincreased by small increments thereafter until the desired or optimumeffect is achieved relative to any negative side effects. Importantdiagnostic measures include those of symptoms of diabetes.

EXAMPLES Example 1 MSC Preparation

MSCs are generated de novo from bone marrow as described in U.S. Pat.No. 5,837,539. Approximately 80-100 ml of marrow was aspirated intosterile heparin-containing syringes and taken to the MDACC Cell TherapyLaboratory for MSC generation. The bone marrow mononuclear cells wereisolated using ficoll-hypaque and placed into twoT175 flask with 50 mlper flask of MSC expansion medium which includes alpha modified MEM(aMEM) containing gentamycin, glutamine (2 mM) and 20% (v/v) fetalbovine serum (FBS) (Hyclone).

The cells were cultured for 2-3 days in 37° C., 5%CO₂ at which time thenon-adherent cells were removed; the remaining adherent cells werecontinually cultured until the cell confluence reached 70% or higher(7-10 days), and then the cells were trypsinized and replaced in sixT175 flasks with MSC expansion medium (50 ml of medium per flask). Asdescribed in Table 5 of U.S. Pat. No. 5,837,539, MSCs isolated andexpanded in this manner are STRO-1 negative.

Example 2 Immunoselection of MPCs by Selection of STRO-3+ Cells

Bone marrow (BM) is harvested from healthy normal adult volunteers(20-35 years old), in accordance with procedures approved by theInstitutional Ethics Committee of the Royal Adelaide Hospital. Briefly,40 ml of BM is aspirated from the posterior iliac crest intolithium-heparin anticoagulant-containing tubes. BMMNC are prepared bydensity gradient separation using Lymphoprep™ (Nycomed Pharma, Oslo,Not⁻way) as previously described (Zannettino, A.C. et al. (1998) Blood92: 2613-2628). Following centrifugation at 400×g for 30 minutes at 4°C., the buffy layer is removed with a transfer pipette and washed threetimes in “HHF”, composed of Hank's balanced salt solution (HBSS; LifeTechnologies, Gaithersburg, Md.), containing 5% fetal calf serum (FCS,CSL Limited, Victoria, Australia).

STRO-3⁺ (or TNAP⁺) cells were subsequently isolated by magneticactivated cell sorting as previously described (Gronthos et al. (2003)Journal of Cell Science 116: 1827-1835; Gronthos, S. and Simmons, P. J.(1995) Blood 85: 929-940). Briefly, approximately 1-3×10⁸ BMMNC areincubated in blocking buffer, consisting of 10% (v/v) normal rabbitserum in HHF for 20 minutes on ice. The cells are incubated with 200 μlof a 10 μg/ml solution of STRO-3 mAb in blocking buffer for 1 hour onice. The cells are subsequently washed twice in HHF by centrifugation at400×g. A 1/50 dilution of goat anti-mouse γ-biotin (SouthernBiotechnology Associates, Birmingham, UK) in HHF buffer is added and thecells incubated for 1 hour on ice. Cells are washed twice in MACS buffer(Ca²⁺—and Mn²⁺—free PBS supplemented with 1% BSA, 5 mM EDTA and 0.01%sodium azide) as above and resuspended in a final volume of 0.9 ml MACSbuffer.

One hundred μl streptavidin microbeads (Miltenyi Biotec; BergischGladbach, Germany) are added to the cell suspension and incubated on icefor 15 minutes. The cell suspension is washed twice and resuspended in0.5 ml of MACS buffer and subsequently loaded onto a mini MACS column(MS Columns, Miltenyi Biotec), and washed three times with 0.5 ml MACSbuffer to retrieve the cells which did not bind the STRO-3 mAb(deposited on 19 Dec. 2005 with American Type Culture Collection (ATCC)under accession number PTA-7282 —see International Publication No.WO2006/108229). After addition of a further 1 ml MACS buffer, the columnis removed from the magnet and the TNAP⁺ cells are isolated by positivepressure. An aliquot of cells from each fraction can be stained withstreptavidin-FITC and the purity assessed by flow cytometry.

Example 3 Cells selected by STRO-3 mAb are STRO-1^(bright) Cells

Experiments were designed to confit. In the potential of using STRO-3mAb as a single reagent for isolating cells STRO-1^(bright) cells.

Given that STRO-3 (IgG1) is a different isotype to that of STRO-1 (IgM),the ability of STRO-3 to identify clonogenic CFU-F was assessed bytwo-colour FACS analysis based on its co-expression with STRO-1⁺ cellsisolated using the MACS procedure (FIG. 1). The dot plot histogramrepresents 5×10⁴ events collected as listmode data. The vertical andhorizontal lines were set to the reactivity levels of <1.0% meanfluorescence obtained with the isotype-matched control antibodies, 1B5(IgG) and 1A6.12 (IgM) treated under the same conditions. The resultsdemonstrate that a minor population of STRO-1bright cells co-expressedTNAP (upper right quadrant) while the remaining STRO-1⁺ cells failed toreact with the STRO-3 mAb. Cells isolated by FACS from all fourquadrants were subsequently assayed for the incidence of CFU-F (Table1).

TABLE 1 Enrichment of human bone marrow cells by dual-colour FACSanalysis based on the co-expression of the cell surface markers STRO-1and TNAP (refer to FIG. 1). FACS sorted cells were cultured understandard clonogenic conditions in alpha MEM supplemented with 20% FCS.The data represents the mean number of day 14 colony-forming cells(CFU-F) per 10⁵ cells plated ± SE (n = 3 different bone marrowaspirates). These data suggest that human MPC are exclusively restrictedto the TNAP positive fraction of BM which co-express the STRO-1 antigenbrightly. Frequency of Enrichment Bone Marrow Fraction CFU-F/10⁵ Cells(Fold Increase) Unfractionated BMMNC  11.0 ± 2.2 1.0 TNAP+/STRO-1bright4,511 ± 185 410 TNAP+/STRO-1dull 0.0 0.0

Example 4 Relative Gene and Surface Protein Expression of Stro-1^(dull)and Stro-1^(bright) Cells

In the first series of experiments, semi-quantitative RT-PCR analysiswas employed to examine the gene expression profile of variouslineage-associated genes expressed by STRO-1^(dull) or STRO-1^(bright)populations, isolated by fluorescence activated cell sorting (FIG. 2A).In the second series of experiments, flow cytometry and mean channelfluorescence analysis was employed to examine the surface proteinxpression profile of various lineage-associated proteins expressed bySTRO-1^(dull) or STRO-1^(bright) populations, isolated by fluorescenceactivated cell sorting.

Total cellular RNA was prepared from either 2×10⁶ STRO-1^(brigt) orSTRO-1^(dull) sorted primary cells, chondrocyte pellets and otherinduced cultures and lysed using RNAzolB extraction method (Biotecx Lab.Inc., Houston, Tex.), according to the manufacturer's recommendations.RNA isolated from each subpopulation was then used as a template forcDNA synthesis, prepared using a First-strand cDNA synthesis kit(Pharmacia Biotech, Uppsala, Sweden). The expression of varioustranscripts was assessed by PCR amplification, using a standard protocolas described previously (Gronthos et al., J. Bone and Min. Res.14:48-57, 1999). Primer sets used in this study are shown in Table 2.Following amplification, each reaction mixture was analysed by 1.5%agarose gel electrophoresis, and visualised by ethidium bromidestaining. RNA integrity was assessed by the expression of GAPDH.

Relative gene expression for each cell marker was assessed withreference to the expression of the house-keeping gene, GAPDH, usingImageQant software (FIG. 2B, C). In addition, dual-colour flowcytometric analysis was used to examine the protein expression profileof ex vivo expanded MPC based on their expression of a wider range ofcell lineage-associated markers in combination with the STRO-1 antibody.A summary of the general phenotype based on the gene and proteinexpression of STRO-1^(dull) and STRO-1^(bright) cultured cells ispresented in Table 3. The data indicate that ex vivo expandedSTRO-1^(bright) MPC exhibit differentially higher expression of markersassociated with perivascular cells, including angiopoietin-1, VCAM-1,SDF-1, TNFα, and RANKL. Comparisons between the protein and geneexpression profiles of STRO-1^(dull) and STRO-1^(bri) cultured cells aresummarised in Tables 3 and 4.

Subtractive hybridization studies were also performed in order toidentify genes uniquely expressed by STRO-1^(bright) cells. Briefly,STRO-1^(dull) and STRO-1^(bright) were isolated as described above (seeFIG. 3A). Total RNA was prepared from STRO-1^(dull) and STRO-1^(bright)cells pooled from 5 different marrow samples using the RNA STAT-60system (TEL-TEST). First-strand synthesize was performed using the SMARTcDNA synthesis kit (Clontech Laboratories). The resultantmRNA/single-stranded cDNA hybrid was amplified by long-distance PCR(Advantage 2 PCR kit; Clontech) using specific primer sites at the 3′and 5′ prime ends formed during the initial RT process according to themanufacturer's specifications. Following Rsal digestion of theSTRO-1bright cDNA, 2 aliquots were used to ligate different specificadaptor oligonucleotides using the Clontech PCR-Select cDNA SubtractionKit. Two rounds of subtractive hybridization were performed usingSTRO-1^(bright) (tester) and STRO-1^(dull) (driver) cDNA, and viceversa, according to the manufacturer's protocol. This procedure was alsoperformed in reverse using STRO-1^(dull) tester cDNA hybridized againstSTRO-1^(bright) driver cDNA.

To identify genes uniquely expressed by STRO-1^(bright) population,STRO-1^(bright)—subtracted cDNA was used to construct replicatelow-density microarray filters comprising 200 randomly selectedbacterial clones transfoiined with the STRO-1^(bri) subtracted cDNAsligated into a T/A cloning vector. The microarrays were subsequentlyprobed with either [³²P1] dCTP—labeled STRO-1^(bri) or STRO-1^(bright)subtracted cDNA (FIG. 3B-C). Differential screening identified a totalof 44 clones, which were highly differentially expressed between theSTRO-1^(dull) and STRO-1^(bright) subpopulations. DNA sequencing of allthe differentially expressed clones revealed that only 1 clone wasrepresentative of a known stromal cell mitogen; namely, platelet-derivedgrowth factor (PDGF) (Gronthos and Simmons, Blood. 85: 929-940, 1995).Interestingly, 6 of the 44 clones were found to contain DNA insertscorresponding to the chemokine, stromal-derived factor-1 (SDF-1). Thehigh abundance of SDF-1 transcripts in human STRO-1^(bright) cells wasconfirmed by semiquantitative RT-PCR of total RNAprepared from freshlysorted STRO-1^(bright), STRO-1^(dull), and STRO-1^(negative) bone marrowsubpopulations (FIG. 3D and Table 3).

TABLE 2 RT-PCR primers and conditions for the specificamplification of human mRNA Target Sense/Antisense Product Gene(5′-3′) Primer Sequences Size GAPDH CACTGACACGTTGGCAGTGG/ 417CATGGAGAAGGCTGGGGCTC SDF-1 GAGACCCGCGCTCGTCCGCC/ 364GCTGGACTCCTACTGTAAGGG IL-1β AGGAAGATGCTGGTTCCCTCTC/ 151CAGTTCAGTGATCGTACAGGTGC FLT-1 TCACTATGGAAGATCTGATTTCTTACAGT/ 380GGTATAAATACACATGTGCTTCTAG TNF-α TCAGATCATCTTCTCGAACC/ 361CAGATAGATGGGCTCATACC KDR TATAGATGGTGTAACCCGGA/ 450 TTTGTCACTGAGACAGCTTGGRANKL AACAGGCCTTTCAAGGAGCTG/ 538 TAAGGAGGGGTTGGAGACCTCG LeptinATGCATTGGGAACCCTGTGC/ 492 GCACCCAGGGCTGAGGTCCA CBFA-1GTGGACGAGGCAAGAGTTTCA/ 632 TGGCAGGTAGGTGTGGTAGTG PPARγ2AACTGCGGGGAAACTTGGGAGATTCTCC/ 341 AATAATAAGGTGGAGATGCAGGCTCC OCNATGAGAGCCCTCACACTCCTC/ 289 CGTAGAAGCGCCGATAGGC MyoDAAGCGCCATCTCTTGAGGTA/ 270 GCGAGAAACGTGAACCTAGC SMMHCCTGGGCAACGTAGTAAAACC/ 150 TATAGCTCATTGCAGCCTCG GFAPCTGTTGCCAGAGATGGAGGTT/ 370 TCATCGCTCAGGAGGTCCTT  NestinGGCAGCGTTGGAACAGAGGTTGGA/ 460 CTCTAAACTGGAGTGGTCAGGGCT SOX9CTCTGCCTGTTTGGACTTTGT/ 598 CCTTTGCTTGCCTTTTACCTC CollagenAGCCAGGGTTGCCAGGACCA/ 387 type X TTTTCCCACTCCAGGAGGGC AggrecanCACTGTTACCGCCACTTCCC/ 184 ACCAGCGGAAGTCCCCTTCG

TABLE 3 Summary of the Relative Gene Expression in STRO-1^(Bri) andSTRO-1^(Dull) populations. A list of genes which displayed measurableand differential expression between the STRO-1^(Bri) and STRO-1^(Dull)populations as determined by reverse transcription-PCR are presented.Values represent the relative gene expression with reference to thehouse-keeping gene, GAPDH. Gene Expression relative to GAPDH STRO- STRO-Tissue Marker 1^(Bri) 1^(Dull) Neurons GFAP (Glial Fibrillary Acidic 0.10.7 Protein) Bone OCN (Osteocalcin) 1.1 2.5 OSX (Osterix) 0.4 1.3 CBFA-1(Core Factor Binding 0.3 0.6 Protein-1) Immunoregulatory RANKL (ReceptorActivator 1.6 0.3 of Nuclear Factor κ B) SDF-1-alpha (Stromal 3.2 0.1Derived factor-1-alpha) Fat Leptin 3.1 4.2 Cardiomyocytes GATA-4 1.1 2.9Enthothelial cells Ang-1 (Angiopoietin-1) 1.5 0.8 Chondrocytes Sox 9 0.31.1 COL X (Collagen X) 3.5 2.8 Pro-inflammatory TNF-alpha (Tumournecrosis 1.7 0.9 Cytokines alpha)

To correlate protein surface expression with density of STRO-1expression, single cell suspensions of ex vivo expanded cells derivedbone marrow MPC were prepared by trypsin/EDTA detachment andsubsequently incubated with the STRO-1 antibody in combination withantibodies identifying a wide range of cell lineage-associated markers.STRO-1 was identified using a goat anti-murine IgM-fluoresceinisothiocyanate while all other markers were identified using either agoat anti-mouse or anti-rabbit IgG-phycoerythrin. For those antibodiesidentifying intracellular antigens, cell preparations were firstlabelled with the STRO-1 antibody, fixed with cold 70% ethanol topermeabilize the cellular membrane and then incubated with intracellularantigen-specific antibodies. Isotype matched control antibodies wereused under identical conditions. Dual-colour flow cytometric analysiswas performed using a COULTER EPICS flow cytometer and list mode datacollected. The dot plots represent 5,000 listmode events indicating thelevel of fluorescence intensity for each lineage cell marker (y-axis)and STRO-1 (x-axis). The vertical and horizontal quadrants wereestablished with reference to the isotype matched negative controlantibodies.

TABLE 4 Summary of the Relative Protein Expression in STRO-1^(Bright)and STRO-1^(Dull) populations. A list of proteins which displayeddifferential expression between the STRO-1^(Bright) and STRO-1^(Dull)populations as determined by flow cytometry are presented. Valuesrepresent the relative mean fluorescence intensity of staining. MeanFluorescence Intensity STRO- STRO- Tissue Marker 1^(Bright) 1^(Dull)Neurons Neurofilament 1.7 20.5 Bone ALK PHOS (Alkaline 5.7 44.5Phophatase) Immunoregulatory RANKL (Receptor Activator 658.5 31.0 ofNuclear Factor κ B) Epithelial Cells CytoKeratin 10 + 13 1.2 23.3Cytokeratin 14 1.8 8.8 Smooth Muscle α-SMA (Alpha Smooth 318.0 286.0Muscle Actin) Chondrocytes Byglycan 84.4 65.9 Basal Fibroblast TenascinC 22.2 6.9 Cardiomyocyte Troponin C 2.5 15.0

These results show that SDF-lalpha and RANKL are highly expressed bySTRO-1^(bright) cells. This is important because both of these proteinsare known to be involved in up-regulation of CD4+ CD25+ regulatory Tcells which confer protection against immune disorders such as GVHD(Loser et al., Nature Medicine 12:1372-1379, 2006; Hess, Biol. BloodMarrow Transplant, 12 (1 Suppl 2):13-21, 2006; and Meiron et al., J.Exp. Medicine 205:2643-2655, 2008).

Example 5 In Vitro Immunosuppressive Activity

To assess immunosuppressive activity of culture-expanded STRO-1^(bright)cells (MPC(B)), we used CD3/CD28 stimulation as a read-out. Results werecompared to a population of culture-expanded, bone marrow-derived STRO-1negative cells isolated as in Example 1 (MSC(A)). Human peripheral bloodmononuclear cells (PBMC) were stimulated with CD3/CD28 coated beads inthe presence of 4 escalating concentrations of MSC and MPC preparations.The proliferation of T cells was measured by 3H-Tdr incorporation.

MSC (A) and Stro-1^(bright) MPCs (B) were tested for their ability tosuppress the response of human peripheral blood mononuclear cells (PBMC)to CD3/CD28 stimulation. MSC and MPC or commercially-purchased controlhuman MSC (Lonza) were added at different ratios to the cultures ofPBMC. After 3 days, 3H-Tdr was added for 18 hours and the cultures thenharvested.

PBMC proliferation in response to CD3/CD28 was inhibited in a dosedependent fashion by all preparations. However, preparation B wasclearly superior to the effect produced by preparation A as well ascontrol hMSC (FIG. 4). At a 1:100 MSC:PBMC ratio, MPC B still inhibited70% of control T cell proliferation, whilst controlcommercially-purchased MSC (Lonza) and MSC A produced a 50% and 60%inhibition, respectively (FIG. 5).

Example 6 In Vivo Effect of MPCs on T Cell Proliferation

For the following experiments the myelin oligodendrocyte glycoprotein(MOG)-induced experimental inflammatory encephalomyelitis (EAE) inC57Bl/6J mice was used. C57Bl/6J mice display similar phenotypicsymptoms (progressive paralysis) to that of MS patients as well asshowing extensive inflammation, demyelination and axonal loss/damage inthe CNS. The immunization procedure for the induction of EAE, assessmentof clinical symptoms and MPC transplantation used is as follows.

Active Induction of EAE

Mice were immunized with 200 μg recombinant MOG dissolved in PhosphateBuffered Saline (PBS) and mixed with an equal volume of Freund'scomplete adjuvant containing 400 μg of killed Mycobacterium tuberculosisH37Ra. 0.1 ml of this mixture was injected subcutaneously into the rightand left flank (total 0.2 ml/mouse) using a 25 gauge (G) needle. Micewere also immunized with 350 ng inactivated Bordetella pertussis toxinin 0.30 ml of PBS intravenously (i.v.) via tail vein of on day 0 and day2 using a 29 G needle. Gentle pressure was applied to the I.V. site for30 sec after the injection to reduce the risk of bleeding from the i.v.site.

Mice were monitored every 2-5 minutes for 10-15 minutes to ensure thereis no active bleeding.

Treatment with MPCs

MPCs were isolated essentially as described in Example 2. On days 8, 10and 12 after disease induction, 2×10⁵ or 4×10⁵ MPCs were administered asa single intravenous (i.v.) injection in a volume of 200 μl PBS (seeTable 5). Controls received i.v. injections of equal volumes of PBSonly. Mice were monitored daily and clinical signs scored according tothe scale described below. Experiments were continued for approximately36 days to monitor the course of disease. At termination of theexperiment, brain, spinal cord and optic nerve were dissected and fixedin formalin solution.

TABLE 5 Summary of Treatment Regimen No of cells Total MPC per mouse perinjected per Treatment injection 20 g mouse Number of mice PBS I.V. — —12 High dose MPC 4 × 10⁵ MPC 6 × 10⁶ MPC/Kg 5 I.V. Low dose MPC 2 × 10⁵MPC 3 × 10⁶ MPC/Kg 5 I.V.

MPC-treated mice and controls were culled on day 36 after diseaseinduction (MOG35-55 immunization). Splenocytes were cultured in vitrowith media alone or re-stimulated with MOG₃₅₋₅₅ and then T-cellproliferative responses were measured through [³H]-thymidineincorporation. The specific proliferative responses to MOG were comparedto the matched splenocytes cultured in media-alone (unstimulated).Splenocytes cultured in PMA/Ionomycin served to determine thenon-specific (antigen-independent) stimulation of T cell proliferation.

Data presented in FIG. 6 demonstrate that T cell immune responses tosecondary in vitro antigenic challenge with MOG are inhibited incomparison to T cells cultured from control animals.

These data show that human MPCs reduce or prevent T cell immune responseto a specific antigen (e.g., antigenic stimulation by MOG), even 24 daysafter the last administration of MPCs. The data indicate that STRO-1enriched MPC induce tolerance to multiple sclerosis antigens.

Example 7 In Vitro Effects of MPCs

The immunoregulatory properties of MPC are tested by proliferationassays, mixed lymphocyte reactions and cytokines production as describedbelow.

Proliferation Assays and Mixed Lymphocyte Reactions

Mononuclear cells are collected from the spleens of healthy C57BL/6mice, 2D2 transgenic mice or MOG-immunized mice treated with MPCs orvehicle alone essentially as described in Example 4. Single cellsuspensions are prepared in complete RPMI media containing 10% FBS, 2 mML-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin (all fromInvitrogen), 1 mM sodium pyruvate (Sigma) and 50 μM β-mercaptoethanol(Sigma). Following red blood cell lysis, cells are washed twice and thenseeded in 96-well flat bottom microtiter plates (Nunc) in triplicate ata concentration of 2.5×10⁵ cells per well in the presence of either 20μg/ml MOG₃₅₋₅₅ (GL Biochem), 800 ng/ml ionomycin and 20 pg/ml phorbolmyristate acetate (PMA) (both from Sigma), or into wells pre-coated with10 μg/ml anti-CD3 and 10 μg/ml anti-CD8 (both from BD). Cells are thenincubated at 37° C. with 5% CO₂ for 72 hours and 1 μCi/well [3H]thymidine is added during the last 18 hours of culture. Cells areharvested onto filter mats and incorporated radioactive nucleic acidscounted on a Top Count Harvester (Packard Biosciences). For experimentsinvolving inhibition of T-cell proliferation by MPC, concentrations ofMPC ranging from 2.5 to 0.002×10⁴ cells per well are seeded prior to theaddition of splenocytes.

In mixed lymphocyte reactions (MLR), 2×10⁵ splenocytes from C57BL/6 mice(responders) are incubated with equal numbers of irradiated (20 Gy)Balb/c stimulators or irradiated MPC and cultured for a period of 5days, with the addition of 1 μCi/well [3H] thymidine during the last 24hours of culture.

In MLRs involving T-cell inhibition, 2×10⁴ irradiated MPC are seededinto the wells prior to the addition of splenocytes.

Cytokine Production

Supernatants used for analysis of cytokine production are obtained fromtwo day co-cultures of 2.5×10⁶ splenocytes from 2D2 transgenic micestimulated with 20 μg/ml MOG₃₅₋₅₅ alone or in the presence of 2×10⁴ MPC(MPC: splenocyte ratio of 1:10). Quantitative analysis of cytokines usperformed using a mouse Th1/Th2/Th17 cytometric bead array (CBA) kit(BD) essentially according to the manufacturer's instructions andanalyzed on a BD FACSCanto II flow cytometer. The following cytokinesare measured: interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17A,interferon-γ (IFN-γ) and tumor necrosis factor-a (TNF-α).

The data of this pilot study have consistently shown thatSTRO-1^(bright) MPCs exhibited superior immunosuppressive capacities ascompared to either no treatment or treatment with STRO-1 negative MSCs.This was evident in the in vitro assay and, most importantly in the invivo assay.

Example 8 Effects of MPCs on PHA-Mediated Lymphocyte Proliferation

PBMC were stimulated with phytohemagglutinin (PHA; 10 ug/ml; SigmaChemical Company, St. Louis, Mo.) to illicit lymphocyte proliferation.STRO-1 bright cells at arrange of concentrations (see Table 6) were ableto significantly suppress PBMC T cell proliferative responses as shownin FIG. 7.

TABLE 6 MLR Dilution of Stro-1^(bri) cells Responder Cell # StimulatorCell (Stro-1^(bri)) # % Stro-1^(bri) cells 50,000/0.1 ml 500/0.1 ml 1%50,000/0.1 ml 2,500/0.1 ml 5% 50,000/0.1 ml 5,000/0.1 ml 10% 50,000/0.1ml 10,000/0.1 ml 20% 50,000/0.1 ml 25,000/0.1 ml 50% 50,000/0.1 ml50,000/0.1 ml 100%

Similar to the human findings ovine MPCs were able to significantlyinhibit levels of alloimmune responses to ovine PBMCs stimulated withPHA (FIG. 8). Ovine STRO-3 selected cells were also able to suppresslymphocyte proliferation in a dose dependent manner when used asstimulators against ovine PBMCs or purified ovine T cells (selectedusing Miltenyi T cell isolation kit) (FIG. 9).

All references cited in this document are incorporated herein byreference.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

1. A method for suppressing T cell activation which comprises contactinga cell population comprising T cells in vitro or ex vivo with aneffective amount of STRO-1⁺ cells and/or soluble factors derivedtherefrom to suppress T cell activation.
 2. A method according to claim2 wherein the cell population is a peripheral blood mononuclear cellsample.
 3. A method according to claim 2 wherein the cell populationcomprise CD25⁺ CD4⁺ T cells of a naïve phenotype (CD45RA⁺).
 4. A methodaccording to any one of claims 1 to 3 which comprises contacting thecell population comprising T cells in vitro or ex vivo with an effectiveamount of STRO-1⁺ cells and/or soluble factors derived therefrom and oneor more factors which induce formation of regulatory T cells.
 5. Amethod according to claim 4 wherein the one or more factors which induceformation of regulatory T cells is selected from the group consisting ofa-melanocyte-stimulating hormone (α-MSH), transforming growth factor-β2(TGF-β2), vitamin D3 and/or Dexamethasone.
 6. A method according to anyone of claims 1 to 4 which further comprises contacting the cellpopulation comprising T cells with one or more agents selected from thegroup consisting of interleukins, antigens, antigen presenting cells,lectins, and antibodies or specific ligands for a cell surface receptorsor combinations thereof.
 7. The method according to claim 6 wherein theinterleukin is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 orcombinations thereof.
 8. A composition of T cells obtained by the methodof any one of claims 1 to
 7. 9. A composition comprising T cells,STRO-1⁺ cells and/or soluble factors derived therefrom, one or morefactors which induce formation of regulatory T cells and apharmaceutically acceptable carrier.
 10. A method for treating anautoimmune disorder in a subject in need thereof comprising treating acell population comprising T cells in vitro or ex vivo with an effectiveamount of STRO-1⁺ cells and/or soluble factors derived therefrom tosuppress T cell activation and administering the treated cells to thesubject.
 11. A method for treating or preventing a disorder caused byexcessive or aberrant T cell activation comprising administering to asubject in need thereof an amount of STRO-1⁺ cells and/or solublefactors derived therefrom effective to suppress T-cell activation in thepatient.
 12. The method of claim 10 or claim 11, comprisingadministering between 0.1×10⁶ to 5×10⁶ STRO-1⁺ cells and/or progenythereof.
 13. The method of any one of claims 10 to 12, comprisingadministering between 0.3×10⁶ to 2×10⁶ STRO-1⁺ cells and/or progenythereof.
 14. The method of any one of claims 10 to 12 comprisingadministering a low dose of STRO-1⁺ cells and/or progeny thereof. 15.The method of claim 14, wherein the low dose of STRO-1⁺ cells and/orprogeny thereof comprises between 0.1×10⁵ and 0.5×10⁶ STRO-1⁺ cellsand/or progeny thereof.
 16. The method of claim 14, wherein the low doseof STRO-1⁺ cells and/or progeny thereof comprises about 0.3×10⁶ STRO-1⁺cells and/or progeny thereof.
 17. A method according to any one ofclaims 10 to 16 which further comprises administering to the subject oneor more agents selected from the group consisting of interleukins,antigens, antigen presenting cells, lectins, and antibodies or specificligands for a cell surface receptors or combinations thereof.
 18. Themethod according to claim 17 wherein the interleukin is IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18 or combinations thereof.
 19. Themethod according to any one of claims 10 to 18, further comprisingadministering an immunosuppressive drug to the subject.
 20. A methodaccording to claim any one of claims 1 to 19 wherein the STRO-1⁺ cellsare enriched for STRO-1^(bright) cells.
 21. A method according to anyone of claims 1 to 20 wherein the STRO-1^(bright) cells and/or progenythereof are allogeneic.